We intend to analyze the role of "muscle activity-induced changes" (i.e. Ca 2 ion and cyclic nucleotide concentrations) in the process of normal mouse muscle-maturation in vitro and contrast such developmental programs with those induced by muscle activity. On the basis of our previous results, we hypothesize that the overall rates of turnover of membrane-associated molecules, such as acetylcholinesterase (ACh E) and acetylcholine recepors (ACh R), as well as the molecular forms of ACh E and specific isozymic forms of the contractile proteins (actin, myosin, myosin light chain kinase) are significantly controlled by muscle activity via ion fluxes, changes in intracellular Ca 2 ion concentrations and levels of cyclic nucleotides; whereas localization of functional synaptic molecules and perhaps even morphologic configurational changes in the muscle motor end plate may be under the direct instructional control of "neurotrophic" molecules. We will attempt to assess the role of membrane activity (Ca 2 ion) changes and alterations in cyclic nucleotide levels, by use of membrane depolarization, variations in internal (Ca 2 ion) by the application of the ionophore A23187 and various extracellular (Ca 2 ion), and by application of permeable cyclic nucleotide derivatives to muscle cultures. Methods of analysis and characterization of muscle maturation, under the specifically controlled conditions, will include ultrastructural examination, assessment of accumulation of contractile proteins by disc gel and two dimensional gel electrophoresis of myofibrillar proteins, isolation and characterization of possible isozymic varients of actin, the light chains of myosin and myosin light chain kinase, study of turnover of ACh R by 125I alpha-Bungarotoxin binding of cultures, and estimates of specific activities, and molecular forms of ACh E by density gradient sedimentation analysis. We hope our results will elucidate some of the molecular mechanisms by which normal membrane activity controls muscle maturation. In addition, we will employ the same type of analysis on mutant dysgenic muscle, which is normally incapable of contraction, to describe the lesion, and to confirm our preliminary results on the mdg "curing" effect of normal neurons.